Biofilms are silent but formidable threats in seafood processing, where Pseudomonas aeruginosa and Escherichia coli can quickly transform contact surfaces into reservoirs of contamination. This study explores the dynamic biofilm formation on aluminum, silicone rubber, stainless steel, and polyethylene terephthalate over 24 and 72 h. Quantitative assays including Colony Forming Unit (CFU), Crystal Violet (CV), 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) revealed a significant increase in biofilm density, particularly on aluminum and silicone rubber. Fourier-Transform Infrared Spectroscopy (FTIR) and 1H Nuclear Magnetic Resonance (NMR) analyses showed that biofilm EPS exhibits an evolving amphiphilic nature, with stable polysaccharides and increasing lipid content enhancing resilience. Confocal Laser Scanning Microscopy (CLSM), and Field Emission Scanning Electron Microscopy (FE-SEM) captured the shift from early attachment to mature, dense biofilms. These findings underscore the crucial impact of surface material on biofilm growth and the pressing need for tailored cleaning protocols to curb contamination risks in food processing environments.